Method for determining 2-amino-5-sulfydryl-1, 3, 4-thiadiazole in vegetables and fruits

Abstract

The invention discloses a method for determining 2-amino-5-sulfydryl-1, 3, 4-thiadiazole in vegetables and fruits, which comprises the following steps of: adding 2-amino-5-sulfydryl-1, 3, 4-thiadiazole into a reaction The alkaline solution is adopted to promote ionization of sulfydryl, so that follow-up derivation is facilitated; the preparation method comprises the following steps: performing sulfydryl protection on an unstable target object by using 1, 4-dithio-DL-threitol, and performing methylation protection on sulfydryl after extraction to form a stable derivative; derivatization is carried out in an alkaline environment by adopting iodomethane, the derivative is accurately qualitative by adopting tandem mass spectrometry, and meanwhile, interference of other impurities on sample quantification is avoided; compared with an existing method, the sensitivity is higher, the quantitation limit is reduced to 4 mu g / kg, and trace residues can be effectively detected.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and particularly relates to a method for determining 2-amino-5-mercapto-1,3,4-thiadiazole in fruits and vegetables. Background Art

[0002] Fungicides such as thiazole zinc, thiazole copper, and thiazole copper are widely used in agricultural disease prevention and control. However, due to their chemical properties, these fungicides are usually insoluble in water and common organic solvents, which poses a technical challenge for their direct quantitative detection. In addition, according to the regulations of GB2763-2021 and GB 2763.1-2022, the main metabolite of these fungicides, 2-amino-5-mercapto-1,3,4-thiadiazole, can be used as a marker for measuring pesticide residues, and it is of great significance to detect it.

[0003] At present, there is no effective detection method for 2-amino-5-mercapto-1,3,4-thiadiazole in China. In October 2022, the "Letter on Soliciting Opinions on the National Standard for Pesticide Residue Detection Methods (Draft for Soliciting Opinions) Nongnong (Pesticide)

[2022] No. 72" was issued, which includes the "Determination of Residues of 3 Pesticides and Their Metabolites Such as Thiazole Copper, Thiazole Zinc, and Thiazole Copper in Plant-derived Foods - High Performance Liquid Chromatography Method (Draft for Soliciting Opinions)". This method uses high performance liquid chromatography to detect the residues of 2-amino-5-mercapto-1,3,4-thiadiazole in plant-derived foods, and calculates the residual levels of thiazole copper, thiazole zinc, and thiazole copper by calculating its content. However, the quantitative limit of the existing detection method is 0.05 mg / kg, which is difficult to meet the limit requirements of thiazole copper in some foods in GB 2763.1-2022. For example, in potatoes, the limit of thiazole copper is 0.01 mg / kg.

[0004] In addition, the 2-amino-5-mercapto-1,3,4-thiadiazole molecule has strong polarity and poor retention effect under reverse phase chromatography conditions. This property further increases the complexity and uncertainty of the detection process. How to break through the bottleneck of the existing technology and develop a detection method with higher sensitivity and higher selectivity to meet the lower limit requirements has become an urgent problem to be solved. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for determining 2-amino-5-mercapto-1,3,4-thiadiazole in fruits and vegetables. The present invention uses a pre-column derivatization method to detect 2-amino-5-mercapto-1,3,4-thiadiazole with a liquid chromatography-mass spectrometry (LC-MS) instrument. Through pre-column derivatization, the unstable mercapto group (-SH) is derivatized into -S-CH3, making the target substance more stable and reducing its polarity, so that it has better retention on the reversed-phase column and is convenient for detection. The derivative is detected by an LC-MS instrument, and the qualitative accuracy is better than that of liquid chromatography, and at the same time, the quantitative limit of the method is reduced to meet the relevant limit requirements of GB 2763-2021 and GB 2763.1-2022.

[0006] The present invention provides a method for determining 2-amino-5-mercapto-1,3,4-thiadiazole in fruits and vegetables, comprising the following steps:

[0007] S1: Add an alkaline buffer solution to the sample, mix well, add an acetonitrile solution, shake and extract, centrifuge, and take out the supernatant;

[0008] S2: Add an alkaline buffer solution and a haloalkane derivatization reagent to the supernatant obtained in S1, derivatize in the dark to obtain a derivatized sample solution;

[0009] S3: Pre-activate the solid-phase extraction column, acidify the derivatized sample solution, transfer it to the pre-activated solid-phase extraction column, wash it successively with water and an acetonitrile solution, squeeze out the water, add an eluent to elute and then filter to obtain a sample to be tested;

[0010] S4: Determine the sample to be tested with a liquid chromatography-tandem mass spectrometry instrument;

[0011] Among them, the alkaline buffer solutions described in steps S1 and S2 contain 0.1-2 m / v% of a mercapto protecting agent.

[0012] The present invention adds a mercapto protecting agent for stabilizing the mercapto group in the target substance 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) to avoid its oxidation and degradation during the pretreatment process. Under alkaline conditions, the mercapto group ionizes to -S-, which can undergo a nucleophilic substitution reaction with haloalkane derivatization reagents such as iodomethane (CH3I) to form methyl thioether (-S-CH3), which has more stable chemical properties and is also more easily ionized and detected.

[0013] In some embodiments, the alkaline buffer solutions described in steps S1 and S2 are any one of a saturated borax solution, a carbonate buffer solution, a phosphate buffer solution, a Tris buffer solution, a glycine-NaOH buffer solution, and a CHES buffer solution.

[0014] In some embodiments, the thiol protecting agent is any one of 1,4-dithiothreitol, 1,4-dithioerythritol, L-cysteine, mercaptoethanol, tris(2-carboxyethyl)phosphine, and sodium thiosulfate.

[0015] In some embodiments, the haloalkane derivative reagent in step S2 is methyl iodide or iodoacetamide.

[0016] In some embodiments, step S1 is specifically as follows: Weigh 0.5 - 5 g of the sample, add 1 - 10 mL of the alkaline buffer solution, mix well, then add 5 - 20 mL of the acetonitrile solution, shake and extract for 5 - 60 min, and take out the supernatant after centrifugation.

[0017] In some embodiments, step S2 is specifically as follows: Take 1 - 20 mL of the supernatant, add 0 - 5 mL of the alkaline buffer solution and 1 - 10 mL of the haloalkane derivative reagent, and perform derivatization in the dark for 5 - 60 min; Adding the buffer solution again in step S2 can prevent the acidic substances in some fruits and vegetables from consuming the alkaline buffer solution, while neutral fruits and vegetables do not need to add the buffer solution again.

[0018] In some embodiments, the chromatographic conditions of the liquid chromatography are as follows: Use a C18 chromatographic column, use an ammonium acetate aqueous solution containing 0.1% formic acid solution as mobile phase A, and use a methanol solution as mobile phase B for gradient elution.

[0019] In some embodiments, the mass spectrometry conditions of the tandem mass spectrometry are as follows:

[0020] Ion source type: electrospray ion source; Ion source temperature: 250 - 650 °C; Nebulization temperature: 250 - 650 °C; Electrospray voltage: 0.5 - 5.5 kV; Nebulizing gas pressure: 0.2 - 0.5 MPa; Auxiliary heating gas: 0.2 - 0.5 MPa.

[0021] In summary, compared with the prior art, the present invention has achieved the following technical effects:

[0022] 1. The present invention uses an alkaline solution to promote the ionization of thiols, thereby facilitating subsequent derivatization; A thiol protecting agent is introduced during the extraction and derivatization processes to effectively protect the thiol structure in 2-amino-5-mercapto-1,3,4-thiadiazole, avoiding its oxidation or degradation in an alkaline environment, and significantly improving the stability of the target substance.

[0023] 2. The present invention performs methylation derivatization of thiols with haloalkanes such as methyl iodide under alkaline conditions to generate stable derivatives, which is conducive to generating high-response characteristic ion signals in liquid chromatography-tandem mass spectrometry (LC-MS / MS), thereby achieving highly sensitive and highly selective detection, and at the same time avoiding interference from other impurities to the quantification of the sample.

[0024] 3. Compared with the existing methods, the detection method of the present invention has higher sensitivity, reduces the quantification limit to 4 μg / kg, and can effectively detect trace residues.

[0025] 4. The method of the present invention is applicable to a variety of fruits and vegetables (such as leafy vegetables, root vegetables, berries, etc.), ensuring the accurate detection of AMT residues in different matrix samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0027] Figure 1 It is the effect diagram of testing 2-amino-5-mercapto-1,3,4-thiadiazole by the liquid phase method of the comparative example of the present invention;

[0028] Figure 2 It is the reaction principle diagram of 2-amino-5-mercapto-1,3,4-thiadiazole and methyl iodide of the present invention;

[0029] Figure 3 It is the liquid chromatography-tandem mass spectrometry diagram of determining 2-amino-5-mercapto-1,3,4-thiadiazole in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0032] (1) Reagents

[0033] Water: meeting the requirements of GB / T 6682; Concentrated hydrochloric acid: analytical pure, 36%-38%; Acetone: analytical pure; Acetonitrile (C2H3N): chromatographic pure; Ammonium acetate: chromatographic grade; Borax (Na2B4O7·10H2O): analytical pure; Ammonium formate: analytical pure; Iodomethane (CH3I, cas number: 74-88-4): 98%; 1,4-Dithiothreitol: biological reagent, BR, ≥98.0%; Saturated borax solution: Heat water, continuously add borax, stir and sonicate to dissolve it, cool the solution to room temperature, filter the precipitated crystals to obtain a saturated solution.

[0034] Borax buffer solution: Weigh 0.5 g of 1,4-dithiothreitol, add 100 mL of saturated borax solution, and dissolve it thoroughly. Prepare it freshly before use.

[0035] Derivatization solution: Pipette 2.5 mL of iodomethane, add acetone to dissolve and make up the volume to 25 mL, and store it in the dark.

[0036] Eluent: Weigh 63 g of ammonium formate, dissolve it in 400 mL of water, add 100 mL of acetonitrile after cooling, and mix well.

[0037] (2) Standard substances

[0038] Standard substance information:

[0039] 2-Amino-5-mercapto-1,3,4-thiadiazole (AMT, cas number: 2349-67-9): ≥99%;

[0040] Preparation of standard stock solution: Weigh an appropriate amount of standard substance, add N,N-dimethylformamide to dissolve and make up the volume to 10 mL.

[0041] Preparation of standard working solution:

[0042] Pipette an appropriate amount of standard stock solution, make up the volume to 10 mL with acetonitrile to prepare a standard working solution of 1 mg / L.

[0043] (3) Instruments and equipment

[0044] Liquid chromatography-tandem mass spectrometer: equipped with an electrospray ionization source; Electronic balance: sensitivity of 0.00001 g and 0.01 g; Vortex mixer; Oscillator; Centrifuge: 4000 r / min; Pipettor: specifications of 20 μL - 200 μL, 50 μL - 1 mL and 1 mL - 5 mL; Centrifuge tube: 50 mL centrifuge tube; Volumetric flask: 25 mL; MCX solid-phase extraction cartridge: cation exchange solid-phase extraction cartridge, 200 mg, 6 cc.

[0045] The specific steps of the method for determining 2-amino-5-mercapto-1,3,4-thiadiazole in fruits and vegetables of this application are as follows:

[0046] (1) Extraction

[0047] Weigh 2 g (accurate to 0.01 g) of the sample, add 5 mL of borax buffer solution, vortex to mix evenly, then add 10 mL of acetonitrile, shake for extraction for 20 min, centrifuge at 4000 rpm for 3 min, take out the supernatant and make up the volume to 20 mL.

[0048] (2) Derivatization

[0049] Accurately pipette 5 mL of the supernatant, add 1 mL of borax buffer solution and 1 mL of derivatizing solution, and perform derivatization in the dark for 30 min.

[0050] (3) Purification

[0051] The MCX solid-phase extraction cartridge is pre-activated with 6 mL of methanol and 6 mL of water. Add 1 mL of 2.4 mol / L hydrochloric acid to the derivatized liquid, transfer all the liquid into the MCX solid-phase extraction cartridge, keep the flow rate below 1 mL / min, wash the cartridge with 6 mL of water and 6 mL of acetonitrile respectively, squeeze out the moisture, accurately add 2 mL of eluent for elution, filter through a 0.22 μm filter membrane, and detect by LC-MSMS.

[0052] (4) Preparation of standard curve

[0053] Respectively pipette 10 μL, 20 μL, 50 μL, 100 μL, 200 μL of the 1 mg / L standard working solution, add 2.5 mL of borax buffer solution and 2.5 mL of acetonitrile, add 1 mL of derivatizing solution, purify according to the steps in (2) and (3), and finally obtain a series of standard solutions with concentrations of 5 μg / L, 10 μg / L, 25 μg / L, 50 μg / L, 100 μg / L.

[0054] (5) Determination

[0055] The liquid phase conditions include:

[0056] Chromatographic column: Eclipse Plus C18 column, 2.1×50 mm, 1.7 μm;

[0057] Mobile phase: Phase A: 4 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid); Phase B: methanol.

[0058] Flow rate: 0.3 mL / min;

[0059] Injection volume: 3 μL;

[0060] Gradient elution program:

[0061] Table 1 Mobile phase gradient

[0062] Time (min) Flow rate (mL / min) Phase A (%) Phase B (%) 0 0.3 95 5 2.0 0.3 5 95 3.5 0.3 5 95 3.51 0.3 95 5 5.0 0.3 95 5

[0063] The conditions of liquid chromatography - tandem mass spectrometry are as follows:

[0064] Ion source: electrospray ionization source; Scanning mode: positive ion scanning; Curtain gas: 35 psi; Collision gas: 9 psi; Ionization voltage: +5500 V; Ion source temperature: 550 °C; Nebulizing gas: 55 psi; Auxiliary heating gas: 55 psi.

[0065] The mass spectrometry parameters of AMT derivatives are shown in Table 2:

[0066] Table 2 Mass spectrometry parameters of AMT derivatives

[0067]

[0068] This method uses the external standard method for quantification. Inject the standard working solution into the liquid chromatography - tandem mass spectrometer, measure the corresponding peak area, plot the standard curve with the peak area as the ordinate and the concentration of the standard determination solution as the abscissa, and calculate the linear regression equation.

[0069] (6) Result calculation

[0070] The content of 2 - amino - 5 - mercapto - 1,3,4 - thiadiazole in the sample is calculated according to formula (1).

[0071]

[0072] In the formula:

[0073] X —— The content of each component in the sample, unit: micrograms per kilogram (μg / kg);

[0074] c —— The concentration of each component in the sample, unit: micrograms per liter (μg / L);

[0075] V —— The volume of sample constant volume, unit: milliliters (mL);

[0076] m —— The mass of the sample represented by the test solution, unit: (g);

[0077] The calculation result is retained to three significant figures.

[0078] (7) Method recovery rate: The recovery rate should be controlled within the range of 60% - 120% to ensure the accuracy of the analysis result.

[0079] (8) Result analysis: Figure 2Schematic diagram of the structural changes of 2-amino-5-mercapto-1,3,4-thiadiazole and its reaction with methyl iodide. Among them, the upper structural formula shows the structural conversion between the keto form (left in the upper figure) and the enol form (right in the upper figure) of 2-amino-5-mercapto-1,3,4-thiadiazole under normal conditions. The lower structural formula shows its reaction process with methyl iodide. The enol form can undergo a nucleophilic addition reaction with methyl iodide in a basic environment, the mercapto group is methylated, and hydrogen iodide is formed, which can continuously consume the enol form and promote the complete conversion of the keto form. Through this derivatization reaction, the response signal and separation effect in the analytical instrument can be improved, and the detection sensitivity and accuracy can be enhanced.

[0080] Figure 3 The first figure in it is the chromatogram of the standard substance at 1 μg / L, the second figure is the chromatogram of the standard substance at 10 μg / L, the third figure is the chromatogram of the blank sample, and the fourth figure is the chromatogram of the quality control sample at 0.4 mg / kg. It can be seen from the chromatograms of the 1 μg / L and 10 μg / L standard substances that under the set liquid chromatography-tandem mass spectrometry conditions, 2-amino-5-mercapto-1,3,4-thiadiazole can be effectively separated, with a stable peak emergence time and a good peak shape, indicating that this method has good selectivity and specificity for the target compound, and has a good linear relationship within this concentration range, and can be used for the quantitative analysis of this compound. In the chromatogram of the blank sample, there are no obvious interfering peaks near the peak emergence time of the target compound, indicating that the blank sample of this method has no interference on the detection of the target compound, proving the reliability of the method.

[0081] Calculated based on the instrument detection limit of 1 μg / L (i.e., 1 ng / mL), it can be detected if there is more than 2 ng in the final eluent (2 mL). From this, it can be deduced that there is at least 2 ng of the target substance in the 5 mL sample taken during derivatization, and there is at least 2 ng × 20 mL / 5 mL = 8 ng in 2 g of the sample; therefore, the calculated detection limit is 8 ng / 2 g = 4 μg / kg.

[0082] Three batches of different samples of Chinese cabbage, apples, and tomatoes were tested using the method of this application, and 2-amino-5-mercapto-1,3,4-thiadiazole (spiked sample) at 0.40 mg / kg was added respectively, and their recoveries were measured.

[0083]

[0084] The results are as shown in Table 3 below and Figure 3 as follows:

[0085] Table 3 Detection Results

[0086]

[0087] The results in Table 3 show that the average recovery rate of the Chinese cabbage sample is 90.17%, that of the apple is 95.24%, and that of the tomato is 94.82%. All of them are within the method recovery rate range of 60% - 120%, indicating that the added 2-amino-5-mercapto-1,3,4-thiadiazole can be accurately recovered and detected, and the method has high accuracy. The relative standard deviation (RSD) of Chinese cabbage is 1.82%, that of the apple is 2.08%, and that of the tomato is 2.67%. The smaller the RSD value, the lower the dispersion degree of the multiple measurement results and the better the repeatability. The RSDs of these three samples are all small, indicating that the detection method of this application has good repeatability in different matrices and the test results are stable and reliable.

[0088] Comparative example

[0089] The 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) in cucumbers was tested by the "National Food Safety Standard - Determination of Residues of Three Pesticides and Their Metabolites, Such as Copper Thiazole, Zinc Thiazole, and Copper Thaisen, in Plant-Derived Foods - Liquid Chromatography Method".

[0090] The sample treatment process is as follows: Accurately weigh 10 g (accurate to 0.01 g) of the test sample into a 50 mL stoppered polypropylene centrifuge tube (add 500 μL of 1 mg / L AMT intermediate solution to the quality control sample), adjust the pH to about 3 with 1 mol / L hydrochloric acid, add 25 mL of acetonitrile, ultrasonically extract for 30 min, vortex extract for 10 min, add 4 g of sodium chloride and then vortex for 2 min, centrifuge at 6500 r / min for 5 min, take out all the supernatant and place it in a pear-shaped flask for rotary evaporation to near dryness, re-dissolve with 1 mL of acetonitrile, fully vortex to dissolve, suck out with a syringe, and pass through a 0.22 μm organic filter membrane for HPLC detection.

[0091] The results are as Figure 1 shown. When testing 2-amino-5-mercapto-1,3,4-thiadiazole by the liquid phase method, the instrument detection limit can only reach 5 mg / L. When the quality control sample is 5 mg / kg, due to the oxidation of the mercapto group in the process, the final detection concentration may be lower than the instrument detection limit and cannot be normally detected.

[0092] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for determining 2-amino-5-mercapto-1,3,4-thiadiazole in fruits and vegetables, characterized in that, It includes the following steps: S1: Add an alkaline buffer solution to the sample. After mixing evenly, add an acetonitrile solution, shake for extraction, and after centrifugation, take out the supernatant. S2: Add an alkaline buffer solution and a haloalkane derivatization reagent to the supernatant described in S1, derivatize in the dark to obtain a derivatized sample solution. S3: Pre-activate a solid-phase extraction column, acidify the derivatized sample solution, transfer it to the pre-activated solid-phase extraction column, wash it successively with water and an acetonitrile solution, squeeze out the moisture, add an eluent for elution and then filter to obtain a sample to be tested. S4: Measure the sample to be tested with a liquid chromatography-tandem mass spectrometry instrument. Among them, the alkaline buffer solutions described in steps S1 and S2 contain 0.1 - 2 m / v% of a thiol protecting agent.

2. The measurement method according to claim 1, characterized in that The alkaline buffer solutions described in steps S1 and S2 are any one of a saturated borax solution, a carbonate buffer solution, a phosphate buffer solution, a Tris buffer solution, a glycine-NaOH buffer solution, and a CHES buffer solution.

3. The measurement method according to claim 1, wherein The thiol protecting agent is any one of 1,4-dithio-DL-threitol, 1,4-dithio-DL-erythritol, L-cysteine, mercaptoethanol, tris(2-carboxyethyl)phosphine, and sodium thiosulfate.

4. The measurement method according to claim 1, characterized in that, The haloalkane derivatization reagent described in step S2 is methyl iodide or iodoacetamide.

5. The measurement method according to claim 1, characterized in that Step S1 is specifically: Weigh 0.5 - 5 g of the sample, add 1 - 10 mL of the alkaline buffer solution, after mixing evenly, add 5 - 20 mL of the acetonitrile solution, shake for extraction for 5 - 60 min, and after centrifugation, take out the supernatant.

6. The measurement method according to claim 1, characterized in that Step S2 is specifically: Take 1 - 20 mL of the supernatant, add 0 - 5 mL of the alkaline buffer solution and 1 - 10 mL of the haloalkane derivatization reagent, and derivatize in the dark for 5 - 60 min.

7. The measurement method according to claim 1, characterized in that, The chromatographic conditions of the liquid chromatography are: Use a C18 chromatographic column, use an ammonium acetate aqueous solution containing 0.1% formic acid solution as mobile phase A, and use a methanol solution as mobile phase B for gradient elution.

8. The measurement method according to claim 1, characterized in that, The mass spectrometry conditions of the tandem mass spectrometry are: Ion source type: electrospray ion source; ion source temperature: 250 - 650 °C; nebulization temperature: 250 - 650 °C; electrospray voltage: 0.5 - 5.5 kV; nebulizing gas pressure: 0.2 - 0.5 MPa; auxiliary heating gas: 0.2 - 0.5 MPa.

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